1. Field of the Invention
This invention relates to methods and apparatus for preservation of specimens by hyper-rapid freezing. More particularly, the present invention relates to methods for hyper-rapid freezing of liquid biological specimens utilizing solid or slushed refrigerants.
2. Description of the Related Art
Freezing has been defined as the solidification of a liquid and may be divided into two kinds: crystallization and vitrification. Crystallization involves an orderly arrangement of molecules, while vitrification is the glass-like solidification of solutions at low temperature without ice crystal formation. Vitrification can be achieved by increasing the viscosity of the solution and by high speeds of cooling and warming. Within certain limits, the higher the speed of the temperature change, the lower the viscosity required to vitrify.
Dubochet, et al. (J. Microscopy, vol. 124, pt. 3, pp. RP3-RP4, 1981) describes novel methods and apparatus for ultra-rapid vitrification of water. Successful vitrification of pure water was achieved by spraying water onto a wire screen and dropping the screen into liquid nitrogen. Microscopic examination of sections of the frozen film of water revealed that the regions near the surface had been vitrified into a noncrystalline glass, although regions where the water film was thicker had frozen into small crystals. Apparently, heat from the water film could be transferred rapidly enough into liquid nitrogen to freeze water into a glass structure before ice crystals had a chance to form. The key to success of the experiment was the high surface to volume ratio (film) of the water, and the rapid decrease in temperature provided by the plunge into liquid nitrogen.
Rapid water vitrification requires extremely rapid freezing rates. Ice crystal size depends primarily on how rapidly heat is removed from the freezing sample. Faster freezing rates lead to smaller ice crystals. If heat energy can be removed quickly enough, then ice crystals will become vanishingly small, and, at fast enough freezing rates, ice crystals will not form at all. Instead, the water molecules slow their movement so quickly they do not have time to form a crystal lattice, and will solidify into a disorganized glass-like structure, i.e. vitrify.
Water molecules are very polarized, and therefore easily form crystal lattices when frozen. Compared to other compounds, the rate of freezing pure water into a vitreous state is extraordinarily rapid on the order of xe2x88x921,000,000xc2x0 F./sec. This freezing rate is so high that in the past, vitrification of pure water was thought to be virtually impossible, and currently only a few technological methods exist that are capable of vitrifying very small quantities of water.
Preservation of biological specimens for future use has long been a goal of the scientific community. Successful methods such as freezing and freeze drying have been achieved for simple biological specimens. Many complex macromolecules and whole cells show reduced activity or viability following freezing or freeze drying by conventional methods.
In general, cell injury or cell death from freezing occurs from one (or both) of two mechanisms. The first mechanism is growing ice crystals, which injure cell membranes and organelles due to their sharp edges. Usually, the ice crystals form and grow primarily in the extracellular space and encroach upon the cell from outside. As freezing progresses, the (relatively) large ice crystals damage or puncture the cell membrane and distort the shape and overall structure of the entire cell. As the freezing process nears completion, intracellular ice formation may also form, with internal ice crystals causing physical damage to organelles and other cellular structures.
The second mechanism is internal cell xe2x80x9cpoisoningxe2x80x9d which results from very high concentrations of intracellular solutes, created by osmotic dehydration. As freezing progresses, extracellular ice crystals form and grow larger. The ice is relatively pure water, and ice crystal growth consumes extracellular liquid water which then increases the extracellular solute concentrations. An osmotic force is created between the intracellular and extracellular spaces which drives water across the cell membrane out of the cell. As extracellular water is consumed by ice growth, it is replaced by intracellular water and the cell becomes osmotically dehydrated. Increasing concentrations of various solutes inside the cell eventually reach toxic levels, damaging or killing the cell.
Both mechanisms operate simultaneously during cell or tissue freezing processes using most current technologies. Balancing the influence of each mechanism to minimize cell damage during freezing and thawing is the goal of any cryopreservation process.
High cooling rates are necessary to freeze biological specimens while avoiding ice crystal formation and maintaining in situ diffusible chemical components. The conventional method is plunging cryoprotected biological specimens into a cooling liquid such as liquid nitrogen. This method may be used in the absence of cryoprotectants if the specimens are thin and mounted on low-mass sample holders and the coolant around the sample is renewed fast enough to prevent the formation of a gaseous layer which would limit heat transfer (Escaig, J. Microscopy, vol. 126, pt 3, pp. 221-229, 1982). Another method in which cryoprotectants are not required is contacting the specimen with a metal block cooled to the temperature of liquid nitrogen or helium (Escaig 1982; Sitte, et al., J. Microscopy, vol. 111, pt 1, pp 35-38, 1977). Problems associated with these methods include the difficulty of making suitable specimen holders and the lack of reproducibility of freezing.
Another approach to preserving biological specimens is freezing under high pressure. Conaway (U.S. Pat. No. 4,688,387) teaches placing the biological specimen into a pressure vessel, applying high pressure to the specimen, and then placing the pressure vessel into liquid nitrogen to achieve cooling. A disadvantage of this method is that long term storage at high pressure and low temperature is not convenient. Therefore, once the specimen is frozen, the pressure is released and the specimen is stored at low temperature. However, the specimen must be placed under high pressure again prior to thawing.
Most protein and peptide specimens exhibit poor survival during the conventional freeze-thaw or freeze-dry processes, with low recoveries of 40% to 90% of the original specimen. This is attributed to physical injury and loss of structural integrity of the protein molecule, presumably from water crystal or osmotic damage during the freezing process.
Cryopreservation agents may be added or controlled freezing rates used in order to maximize cell survival. Cryopreservation agents, such as DMSO or propylene glycol, reduce the freezing temperature point and ice crystal size, and at optimal concentrations the cryopreservation agents will significantly increase cellular freeze-thaw survival. However, excessive concentrations of cryopreservatives are toxic to cells and tissue, limiting their usefulness. Controlled freezing rates allow better optimization of the two cell damage mechanisms at different times during the freezing process, and use of plateau and stopping points to permit ice crystal seeding further enhances cell survival for some processes. Optimal freeze-thaw survival for some current applications requires a complex process of cell preparation, changing concentrations of cryopreservation agents, multi-step freezing rates, multiple ice crystal seedings, and finally a rapid plunge into liquid nitrogen or other cryogenic liquefied gas to vitrify the remaining liquid that had not already frozen into ice crystals. Complex thawing protocols may also be required to minimize ice crystal or hyper-concentrated solutes from further damaging the cells.
Several types of biological specimens have been successfully frozen and thawed using cryopreservatives and various freezing rates. Examples include plant material, tissue culture cells, sperm and embryos. Oocytes, however, are particularly difficult to cryopreserve for the following reasons:
1) High volume-to-surface ratio. Oocytes are the largest cells of the mammalian body. During equilibration, high concentration gradients of cryoprotectants may arise resulting in toxic damage in one part of the cell and insufficient protection in the other part of the cell. Osmotic effects are also increased and during equilibration and dilution oocytes may suffer extreme changes of shape resulting in possible damages of membranes and cytoskeleton.
2) Chilling injury. In bovine oocytes, low temperature sensitivity is attributed to two different factors. Besides damage of the numerous lipid droplets, the meiotic spindle of the metaphase II oocytes suffers serious injury when cooled to 4 or even 25xc2x0 C. Unlike the lipid alteration, damage to the spindle after short-time low-temperature exposure is reversible, but may even so disturb the subsequent fertilization process.
3) Alteration of the zona pellucida. Cooling may result in decreased fertilization rates caused by premature cortical granule exocytosis and zona hardening.
4) Cumulus cell removal. Both immature and mature oocytes form a functional unit with the attached cumulus cell layers. Unlike the in vivo process, the cumulus investment is beneficial for in vitro fertilization either by promoting sperm capacitation or preventing polyspermy. However, these layers and the accumulated glycoproteins reduce the speed of cryoprotectant penetration either at equilibration or at dilution. In a group of oocytes, partial removal of cumulus cells is very difficult to perform uniformly, and the differences may result in uneven cryoprotectant penetration (Vajta, IETS Newsletter, June 1997, http://www.iets.uiuc.edu/news/iets697.html#featured). The damage sustained is often enough to kill the cell or prevent if from successfully fertilizing.
Martino, et al (Biology of Reproduction, vol. 54, pp 1059-1069, 1996) discloses a method for cryopreserving bovine oocytes in which oocytes were placed in a cryopreservative medium, and placed either in straws or on electron microscope grids. The straws were plunged into liquid nitrogen and the grids were either plunged into liquid nitrogen or nitrogen slush. Martino, et al. report that cooling rates achieved with grids were much higher than with straws. To test whether the use of even faster cooling rates would improve survival, nitrogen slush was compared to liquid nitrogen for freezing oocytes on grids. Survival rates based on morphology, cleavage and blastocyst formation were higher for oocytes frozen in liquid nitrogen compared to those frozen in nitrogen slush.
Successful cryopreservation of biological specimens without the risk of cryoprotectant toxicity and loss of viability common with current methods would have significant clinical and scientific applications. Successful cryopreservation of human oocytes would have significant clinical applications, including frozen quarantine of donor eggs to prevent HIV or hepatitis transmission to the recipient, long-term storage of oocytes for chemotherapy patients who wanted to later attempt pregnancy, or for women undergoing oophorectomy for benign disease, or for short-term storage for women with ovarian hyper-stimulation syndrome.
The practice of freezing and storing sperm and embryos has achieved a certain amount of success. Freezing and thawing unfertilized oocytes, however, has not generally been successful for reasons set forth above. The ability to freeze and store oocytes and sperm separately would avoid the legal complications regarding custody and maintenance of frozen embryos. The instant invention is particularly useful for freezing unfertilized oocytes.
An embodiment of the instant invention is a method of rapidly freezing and storing water or liquid samples without the use of cryoprotectants to minimize or eliminate damage from ice crystals or osmotic forces, allowing very high recovery rates upon thawing. The liquid sample is transformed into very small drops which are contacted directly with a refrigerant which is at least partially solidified. The refrigerant may be a solid undergoing a solid-to-liquid phase change, or a solid-to-gas phase change. This method is especially useful for substances that are susceptible to ice crystal or osmotic damage, such as proteins, peptides, and other macromolecules.
A further embodiment of the invention is a method of stopping a chemical or biological reaction by rapidly freezing the reaction mixture through direct contact with a refrigerant which is at least partially solidified. Molecular slowing occurs almost instantly, preserving the compounds in mid-reaction. The various molecular steps of the reaction are frozen in place, and can be analyzed by studying the vitrified sample.
A further embodiment of the invention is to provide a method for lyophilizing chemical and biological compounds or compositions while retaining structural integrity. Reconstitution of the substance will restore the original liquid sample with little or no damage. Specific applications of this type of lyophilization include freeze dried foodstuffs (coffee, milk, juices, etc.), medical supplies (vaccines, antibiotics, etc.) and laboratory processes (protein extractions, molecular biology samples, etc.).
An additional embodiment of the invention is a method for production of fine-grained or amorphous solids by direct deposition of a vapor substance onto a cryogenic gas which is at least partially solidified.
Another embodiment of the invention is an apparatus for rapid freezing a liquid sample. The apparatus comprises a means for driving the liquid sample directly onto a refrigerant which is at least partially solidified. Another embodiment of the apparatus is one which further comprises a means for transforming the liquid sample into very small drops prior to driving them onto the refrigerant.
A method was developed for rapidly freezing a sample in which primary and secondary refrigerants in thermal contact are used to achieve the state of at least partial solidification of the primary refrigerant. Another method for achieving a state of at least partial solidification of the primary refrigerant is evaporative cooling by placing the refrigerant in a vacuum chamber and subjecting it to low pressure.
Various additional features can be included in further embodiments of the invention. One such feature is an apparatus with multiple freezing chambers. Another feature is an apparatus with a continuously renewed solidified primary refrigerant surface. Additional features include apparatus for direct injection of sample onto solid or into partially solidified refrigerant.
An additional embodiment of the invention is a method for rapidly freezing non-liquid samples by contacting them directly with a refrigerant which is at least partially solidified.